Process for the preparation of autocrosslinked compounds of hyaluronic acid and the derivatives thereof by the supercritical antisolvent technique

ABSTRACT

Crosslinked compounds of hyaluronic acid and the derivatives thereof, prepared by the technique of precipitation induced by supercritical antisolvent (SAS), can be used to advantage for the preparation of biomaterials for use in the field of medicine and surgery and in tissue engineering for medical and surgical purposes.

This application is the national phase under 35 U.S.C. 371 of PCTInternational Application No. PCT/EP00/02814 which has an Internationalfiling date of Mar. 30, 2000, which designated the United States ofAmerica and was published in English.

SUBJECT OF THE INVENTION

The present invention describes a process for the preparation ofautocrosslinked compounds of hyaluronic acid and the derivatives thereofby the technique of precipitation induced by supercritical antisolvent(SAS).

These crosslinked compounds of hyaluronic acid and the derivativesthereof can be used to advantage for the preparation of biomaterials foruse in the field of medicine and surgery and in tissue engineering formedical and surgical purposes.

BACKGROUND OF THE INVENTION

Hyaluronic acid is a heteropolysaccharide composed of alternatingresidues of D-glucuronic acid and N-acetyl-D-glycosamine. It is astraight-chained polymer with a molecular weight that varies between50,000 and 13,000,000 Da according to, the source from which it isobtained and the methods of preparation and determination used. It isnaturally present in the pericellular gels, in the fundamental substanceof connective tissue and in vertebrate organisms, of which it is one ofthe chief components, in the synovial fluid of joints, in the vitreoushumor, in the human umbilical cord tissues and in rooster combs.

Hyaluronic acid plays a vital role in many biological processes such astissue hydration, proteoglycan organisation, cell differentiation,proliferation and angiogenesis (J. Aigner et al., L. Biomed. Mater. Res.1998, 42, 172-181).

It is known that hyaluronic acid fractions can be used to facilitatetissue repair, as substitutes for the intraocular fluid, or they can beadministered by the intra-articular route to treat joint pathologies, asdescribed in European Patents Nos. 0138572 and 0535200.

Hyaluronic acid plays a fundamental role in the tissue repair process,especially in the early stages of granulation, stabilising thecoagulation matrix and controlling its degradation, favouring therecruitment of inflammatory cells such as polymorphonucleate leukocytesand monocytes, of mesenchymal cells such as fibroblasts and endothelialcells and, lastly, orientating the successful migration of epithelialcells.

It is known that the application of hyaluronic acid solutions is able toaccelerate healing in patients suffering from sores, wounds and burns.The role of hyaluronic acid in the various stages of the tissue repairprocess has been described by the construction of a theoretical model byWeigel, P. H. et al.: “A model for the role of hyaluronic acid andfibrin in the early events during the inflammatory response and woundhealing”, J. Theor: Biol:, 119, 219, 1986.

The use of low-molecular-weight hyaluronic acid fractions for thepreparation of pharmaceutical compositions with bone-inducing properties(U.S. Pat. No. 5,646,129) is also known.

Hyaluronic acid derivatives maintain all the properties of the aboveglycosaminoglycan, with the advantage that they can be processed invarious forms and that their solubility and degradation times can bevaried according to the percentage of their derivation (EP 0216453 B1).

Moreover, also known are the total or partial esters of hyaluronic acidand the autocrosslinked derivatives of hyaluronic acid, their use in thepharmaceutical and cosmetic fields and in that of biodegradablematerials (U.S. Pat. Nos 4,851,521; 4,965,353; 5,676,964).

The process known to date for the preparation of autocrosslinkedderivatives of hyaluronic acid has the disadvantage that it is difficultto achieve homogeneous autocrosslinking throughout the mass of theproduct. Indeed, said process consists in preparing a solution ofhyaluronic acid salt with tetrabutylammonium (HA-TBA) in an organicsolvent such as N-methyl-pyrrolidone (NMP), dimethylsulfoxide (DMSO) ordimethylformamide (DMF) at a temperature of below 0° C., agitatingconstantly while a solution of 2-chloro-1-methyl-pyridinium iodide(CMPJ) is added to whichever solvent or organic solvent is being used,the quantity of crosslinking agent being 25% that of the polymer mass.As soon as contact is made, the CMPJ produces crosslinking in the HA-TBAand, as its viscosity increases the solid becomes segregated before thecrosslinking agent is homogeneously distributed. It therefore tends tosettle preferentially on the solid and is no longer able to penetrateinto the solution, leading to uneven crosslinking.

The ability of antisolvent fluids such as carbon dioxide (CO₂) tosolubilise in organic solvents under increased pressure has beenexploited, in the present invention, to try to control the crosslinkingreaction which is characterised by very rapid kinetics, especially inthe early stage of mixing the reagents.

Rendering liquids far less viscous by swelling them makes it possible toachieve a mixture more rapidly and more efficiently than by thetraditional process.

By compressing the antisolvent fluid it is possible to work in aswollen, and therefore dispersed environment and to control thecrosslinking reaction once it has begun, slowing it down.

Addition of the crosslinking agent to the swollen mass is very rapidbecause of a specially designed injection system. Because of theswelling caused by the compressed antisolvent, the agitation of the massand the fast injection of the crosslinking agent, it is possible tocontrol the rate of crosslinking at the onset at temperatures of between0 and 20° C., preferably 10° C.

At this temperature, the viscosity of the medium is such as will allowgood homogenisation of the mass, unlike in the case of the conventionalprocess that has to be conducted at temperatures of around −20° C.

It is essential to work below precipitation pressure so that theseparation of the polymer from the liquid phase occurs through theeffect of the crosslinking and is not induced by precipitation due tothe antisolvent effect; in the latter case, the crosslinking agent isonly able to trigger the reaction superficially because it is unable topenetrate within the precipitate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a process for the preparation ofautocrosslinked compounds of hyaluronic acid and the derivatives thereofby means of precipitation using the supercritical antisolvent (SAS)technique.

These crosslinked compounds of hyaluronic acid and the derivativesthereof can be used to advantage for the preparation of biomaterials foruse in the medical and surgical fields and in tissue engineering in themedical-surgical field.

BRIEF DESCRIPTION OF DRAWINGS

The equipment used the technique is illustrated in FIG. 1, whereinP=precipitator; PI=CO₂ pump; P2=pump for the crosslinking agent; VM1,VM2=millimetric valves for flow adjustment; VNR=cut-off valve; V1=on-offvalve; VT1, VT2=three-way valves; A=sampling loop; PE=post-expansionvessel; R=rotameter.

The process according to the present invention involves the followingsteps:

a) preparing solutions of quaternary ammonium salt of hyaluronic acid orthe derivatives thereof and the crosslinking agent;

b) loading the solutions of quaternary ammonium salt and crosslinkingagent into the precipitator and into the container respectively, thelatter being fitted with a pump to suck up the liquid;

c) adjusting the rotation speed to within a range of 200-1000 rpm,preferably between 250 and 450 rpm;

d) adjusting the temperature to within a range of −20° C. to 20° C.,preferably between 0 and 10° C.;

e) adjusting the pressure of the fluid to below that of precipitation ofthe quaternary ammonium salt of hyaluronic acid or the derivativesthereof, which depends upon the solvent and the temperature;

f) injecting the solution of crosslinking agent into the precipitator ata greater pressure than that present in the precipitator in order todisperse all the crosslinking agent homogeneously in the startingpolymer solution;

g) optionally, injecting a buffer solution into the precipitator;

h) increasing the temperature to+ between 20 and 50° C., preferablybetween 15 and 40° C.;

i) leaving it to react for between two and twelve hours, preferably sixhours;

j) washing the product with a flow of antisolvent at a pressure ofbetween 60 and 150 bar, preferably between 80 and 100 bar;

k) depressurising and harvesting the product.

The following hyaluronic acid derivatives are to be preferred:

partial esters of hyaluronic acid wherein part of the carboxy functionsare esterified with alcohols of the aliphatic, aromatic, arylaliphatic,cycloaliphatic, heterocyclic series (EP 0216453 B1);

partial esters of hyaluronic acid wherein part of the carboxy functionsare esterified with an arylaliphatic alcohol and the second part withlong-chain, straight aliphatic alcohols with between 10 and 22 carbonatoms (WO 98/08876)

the partially O-sulfated (WO 95/25751) and/or N-sulfated derivatives (WO98/45335);

the amide derivatives of hyaluronic acid.

The preferred quaternary ammonium salts are tetrabutylammonium salts.The crosslinking agents are, for example, 2-chloro-1-methyl-pyridiniumiodide, 2-chloro-pyridine, 2-chloro-1-isopropyl-pyridinium iodide,1-fluoro-2,4-dinitrobenzene.

The solutions are prepared in organic solvents such asN-methyl-pyrrolidone (NMP), dimethylsulfoxide (DMSO), dimethylformamide.

EXAMPLE

Preparation of crosslinked hyaluronic acid starting from solutions ofhyaluronic acid salts with tetrabutylammonium

The preparation of autocrosslinked hyaluronic acid was conductedstarting from solutions of hyaluronic salts with tetrabutylammonium(HA-TBA; the preparation of these salts is disclosed in EP 216.453, EP342.557 and EP 535.200) in N-methylpyrrolidone (NMP) at a concentrationof 15 and 20 mg/ml at two different temperatures. The working conditionsduring the tests are reported in Table 1.

TABLE 1 Working conditions during the crosslinking tests on a solutionof HA-TBA in NMP with the addition of triethylamine to counteract theacidity produced by the reaction Conc. test (mg/ml V (ml) T (° C.) P(bar) 4 15 100 0 15 5 20 100 10 20

Working Procedure

The solutions containing the polymer and the crosslinking agent areprepared and then loaded into the precipitator and the containerconnected to the pump, respectively.

The precipitator is made airtight and connected to the supply and outletsystem of the apparatus, as well as to the solvent injection line. Amechanical stirrer is then fitted and set at a speed of 400 r.p.m..

The temperature inside the precipitator is maintained at a value withinthe range of 0 to 20° C., preferably 0° C.:

Once the desired temperature has been reached, loading of theprecipitator can begin.

When the pressure has reached the desired value, lower than thatrequired for precipitation, the flow is closed off with the VM1 valveand the system is kept under agitation at a constant pressure.

At this point the pump is activated and begins to suck up the solutioncontaining CMPJ from a tank and force it into the injection line. Byoperating the three-way valves VT1 and VT2, the flows are switched overso as to place lines I and II in communication with the CO₂. Thepressure of the line is adjusted to a higher value than that in theprecipitator, so as to guarantee conditions for the injection of theentire quantity in the loop. At this point, the VI valve is turned on,allowing the solution with the crosslinking agent to be injected intothe pressurised container. This step lasts a fraction of a second, andthen the VI valve is shut off.

Keeping the system under stirring, the temperature of the fluid israised to within a range of 20 to 50° C., preferably 35° C., while theprecipitator is emptied to keep the pressure value below that requiredfor precipitation.

Stirring is continued for a period that may vary between 4 and 12 hours,preferably 6 hours, at the desired temperature, to allow completecrosslinking to be achieved by the crosslinking agent, without anyprecipitation of the solute due to the antisolvent effect.

The precipitation vessel is depressurised by means of the VM2 valve.

The system is left for a few minutes in these conditions, and then theagitation speed is lowered to a value of between 50 and 500 rpm,preferably 200 rpm, and depressurisation is begun by means of the VM2valve. Once the precipitator has been emptied of all the CO₂, it isdisconnected from the crosslinking line, which is cleaned with a flow ofpure NMP fed through the same pump.

The vessel is then disconnected from the line and the crosslinkedpolymer is harvested.

The invention being thus described, it is clear that these methods canbe modified in various ways. Such modifications are not to be consideredas divergences from the spirit and purpose of the invention and any suchmodification that would appear evident to an expert in the field comeswithin the scope of the following claims.

What is claimed is:
 1. Process for the preparation of autocrosslinkedcompounds of hyaluronic acid and derivatives thereof by the technique ofprecipitation induced by antisolvent, comprising the following steps: a)preparing solutions of the quaternary ammonium salt of hyaluronic acidor derivatives thereof and a crosslinking agent; b) loading thesolutions of quaternary ammonium salt and crosslinking agent containerbeing fitted with a pump to suck up the liquid; c) adjusting therotation speed to within a range of 200-1000 rpm; d) adjusting thetemperature to within a range of −20 to 20° C.; e) adjusting thepressure of the fluid to below that required for precipitation of thequaternary ammonium salt of hyaluronic acid or derivatives thereof; f)injecting the solution of crosslinking agent into the precipitator at apressure that is greater than that in the precipitator; g) optionally,injecting a buffer solution into the precipitator; h) increasing thetemperature to a value within a range of 20 to 50° C.; i) leaving it toreact for between 2 and 12 hours; j) washing the product with a flow ofantisolvent at a pressure of between 60 and 150 bar; k) depressurizingand harvesting the product.
 2. Process according to claim 1, wherein thehyaluronic acid derivatives are the partial esters of hyaluronic acidwherein part of the carboxy functions are esterified with alcohols ofthe aliphatic, aromatic, arylaliphatic, cycloaliphatic, heterocyclicseries.
 3. Process according to claim 1, wherein the hyaluronic acidderivatives are the partial esters of hyaluronic acid wherein part ofthe carboxy functions are esterified with an araliphatic alcohol and thesecond part with straight, long-chain aliphatic alcohols with between 10and 22 carbon atoms.
 4. Process according to claim 1, wherein thehyaluronic acid derivatives are partially O-sulfated and/or N-sulfatedderivatives.
 5. Process according to claim 1, wherein the hyaluronicacid derivatives are the amide derivatives of hyaluronic acid. 6.Process according to claim 1, wherein the quaternary ammonium salt istetrabutylammonium salt.
 7. Process according to claim 1, wherein thecrosslinking agents are chosen from the group consisting of2-chloro-1-methyl-pyridinium iodide, 2-chloro-pyridine,2-chloro-1-isopropyl-pyridinium iodide, 1-fluoro-2,4 dinitrobenzene. 8.Process according to claim 1, wherein the solutions are prepared in anorganic solvents chosen from the group consisting ofN-methyl-pyrrolidone (NMP), dimethylsulfoxide (DMSO), dimethylformamide.9. The process according to claim 1 wherein in the step c), saidrotation speed is adjusted within a range of 250-450 rpm.
 10. Theprocess according to claim 1 wherein in the step d), said temperature isadjusted within a range between 0-10° C.
 11. The process according toclaim 1 wherein in the step h), said temperature is increased to a valuewithin a range between 20-40° C.
 12. The process according to claim 1wherein in the step i), said reaction is lasted for 6 hours.
 13. Theprocess according to claim 1 wherein in the step j), said pressure isbetween 80-100 bar.
 14. Process for the preparation of autocrosslinkedcompounds of hyaluronic acid and derivatives thereof by the technique ofprecipitation induced by antisolvent, comprising the following steps: a)preparing solutions of the quaternary ammonium salt of hyaluronic acidor derivatives thereof and a crosslinking agent; b) loading thesolutions of quaternary ammonium salt and crosslinking agent containerbeing fitted with a pump to suck up the liquid; c) adjusting therotation speed to within a range of between 250 and 450 rpm; d)adjusting the temperature to within a range of −20 to 20° C.; e)adjusting the pressure of the fluid to below that required forprecipitation of the quaternary ammonium salt of hyaluronic acid orderivatives thereof; f) injecting the solution of crosslinking agentinto the precipitator at a pressure that is greater than that in theprecipitator; g) optionally, injecting a buffer solution into theprecipitator; h) increasing the temperature to a value within a range ofbetween 15 and 40° C.; i) leaving it to react for between 2 and 12hours; j) washing the product with a flow of antisolvent at a pressureof between 60 and 150 bar; k) depressurizing and harvesting the product.